Toll-like receptors (TLRs) and complement are two components of innate immunity that are critical for first line host defense and elicitation of adaptive immune responses. We have recently uncovered a profound regulatory effect of complement activation on TLR signaling in vivo. The long-term objective of this proposal is to understand the nature and mechanism of interaction between the complement and the TLR systems and to apply such knowledge to the benefit of preventing and treating human infectious, inflammatory and autoimmune diseases. Our preliminary data demonstrated that simultaneous complement activation and TLR ligation in mice greatly potentiated TLR-dependent production of the inflammatory cytokines TNF-?, IL-6, IL-10 and IL-1? and/or diminished the production of IL-12. This regulatory effect of complement on TLR responses was observed in several TLRs examined so far and correlated with increased MAP kinase and NF-?B signaling in the case of TLR4 activation by LPS. We further demonstrated that the altered inflammatory cytokine profile by complement potently induced Th-17 cell differentiation in an in vitro assay. To extend these studies and to understand the mechanism and implications of the cross talk between these two central innate immune systems, we propose the following four specific aims: Specific aim 1 To determine the specificity of the interaction between complement and TLRs. Does complement regulate the activation of all TLRs? With a given TLR, is the effect of complement ligand-specific? Does complement regulate both MyD88-dependent and - independent pathways of TLR activation? Specific aim 2 To determine the specificity and site of action of DAF as a membrane regulator in inhibiting LPS-induced complement activation in vivo. Specific aim 3 To identify the complement mediators that exert the regulatory effect and determine their mechanisms of action. We hypothesize that the complement effect is mediated by the anaphylatoxins C5a and C3a and that there is a convergence of TLR and C5a/C3a mediated intracellular signaling. Specific aim 4 To test the hypothesis that complement-dependent augmentation in pro-inflammatory cytokine production enhances Th-17 cell differentiation in vitro and in vivo. These studies will shed light on the synergistic interaction between two essential arms of the innate immune system and generate new knowledge relevant to inflammation, autoimmunity and vaccine development. PUBLIC HEALTH RELEVANCE: Complement and Toll-like receptors (TLRs) are two forms of the body's natural immune system that defend the host by producing inflammatory mediators and by priming the body's specific immune system called T cells and B cells. When dysregulated, these two systems could also produce tissue injury, leading to pathogenesis of autoimmune diseases. So far, TLR and complement have been studied mostly as independent systems and not much is known about their interaction in host defense and autoimmunity. The focus of this proposal is to characterize a regulatory role of complement on TLR signaling that we have recently discovered. The proposed studies will shed new light on the synergistic interaction between these two important natural immune systems and have relevance to the treatment of human infectious, autoimmune and inflammatory diseases.